WO2006032232A2

WO2006032232A2 - Novel lysolipin biosynthesis gene cluster

Info

Publication number: WO2006032232A2
Application number: PCT/DE2005/001494
Authority: WO
Inventors: Wolfgang Wohlleben; Stefan Pelzer; Patricio Lopez; Andreas Hornung
Original assignee: Combinature Biopharm Ag
Priority date: 2004-09-24
Filing date: 2005-08-22
Publication date: 2006-03-30
Also published as: WO2006032232A3; DE112005002934A5

Abstract

The invention relates to novel genes from lysolipin-producing microorganisms, to novel gene clusters formed from novel and/or known genes of these microorganisms, to expression products of these genes, to a transformation vehicle containing these genes, gene fragments or gene clusters, to transformed cells containing these genes or gene clusters, to methods for the biotechnological production of lysolipin derivatives, to methods for the combinatory biosynthesis of lysolipin derivatives, to novel lysolipin derivatives, to uses of these lysolipin derivatives, and to pharmaceutical compositions containing these lysolipin derivatives.

Description

New lysolipin biosynthetic gene cluster

Field of the invention:

The invention relates to a novel lysolipin biosynthetic gene cluster, new genes or gene fragments from such a biosynthetic gene cluster,

Expression products of these genes or gene fragments, transformation vehicles containing such genes, gene fragments or gene clusters, transformed cells containing such genes, gene fragments or gene clusters, methods for the biotechnological production of lysolipin and lysolipin derivatives, methods for combinatorial biosynthesis of lysolipin derivatives, new

Lysolipin derivatives, uses of such novel lysolipin derivatives and pharmaceutical compositions contain such lysolipin derivatives.

Background of the Invention and Related Art:

The bacterial strain Streptomyces tendae TÜ4042 (CB28) produces the polyketide antibiotic lysolipin, a polycyclic xanthone with strong antibiotic activity against bacteria and antitumor activity against various tumor cell lines. In 1975, lysolipin was first described as a metabolite of the strain Streptomyces violaceon Tü96 (Drautz et al., 1975, Arch Microbiol. 106, 175-190). The term lysolipin actually describes two substances. The lysolipin X secreted by the producer strain is converted to the more stable lysolipin I by light, heat or UV radiation (FIG. 1). Lysolipin I has a 10-50-fold higher biological activity. The antibacterial effect of lysolipin I presumably consists in an inhibition of cell wall biosynthesis by interaction with the carrier lipid, since murein precursors are increasingly accumulated after lysolipin addition in the inhibited cells. Lysolipin I is very effective against Gram-positive (MIC 0.001 μg / ml), as well as against some Gram-negative bacteria. Furthermore, a strong tumorstatische effect against different Tumor cell lines (IC ₅₀ 0.001 ug / ml) are detected (Pultar, 1988, Disseration University of Tübingen).

The biosynthesis of lysolipin has been studied by means of incorporation studies of radioactively labeled precursors (Bockholt et al., 1994, J. Org. Chem. 59, 2064-2069). In these studies, it was found that the synthesis of lysolipin from 12 malonyl CoA units follows a typical polyketide synthesis scheme of type II (characteristic of aromatic polyketides). Rather unusual is the use of malonyl CoA as a starter unit which is complete, i. without decarboxylation. So far, there are only a few known substances that use malonyl-CoA as a starter, such as tetracycline and cycloheximide. Comparing the biosynthesis of these two antibiotics with that of lysolipin, lysolipin exhibits "reverse" orientation of the malonate molecule, and the nitrogen-containing heterocycle of lysolipin is believed to be formed through the intermediary of a malonamide intermediate

Biosynthesis of the backbone numerous modifications must take place to produce the final product lysolipin X or I. These include cyclization and aromatization, introduction of oxygen atoms (9 of the 12 oxygen atoms in lysolipin X are probably introduced from molecular oxygen by oxygenases, including both xanthone oxygens), chlorination presumably by a halogenase, and introduction of methyl groups on the hydroxy groups (C6, C16 , C24), methylene group C28 and nitrogen, presumably by methyltransferases.

Due to its structure, lysolipin can be assigned to the substance class of N-containing polycyclic xanthones. Other related structures of bacterial origin with pharmaceutically interesting properties are the antibiotics albofungin / Chloralbofungin, cervinomycin, simaomicin, citreamicin, the cytostatic and antifungisch Actinoplanone and the antifungisch active substances Seh 42137 and Seh 54445. The pharmaceutical use of these substances has hitherto been prevented by the poor solubility and the toxicity anticipated on account of the xanthone structure and proven for some substances. However, chemical modification has succeeded, after acetylation of cervinomycin A1, in producing derivatives that are more soluble, less toxic, and even more active (US 4,886,884). The possibilities of chemical variation are limited by the presence of "natural protecting groups", as in the highly modified lysolipin.

Compared with the substances known to date, improved pharmacological properties, for example also reduced toxicity, as well as higher selectivity and specificity, are desirable. The derivatization required for this is readily, if not impossible, for the reasons given above.

Technical problem of the invention:

The invention is therefore based on the technical problem of providing means for the synthesis of novel lysolipin derivatives, in particular of lysolipin derivatives which are difficult or difficult to access and which also have improved pharmacological properties.

Broad features of the invention and embodiments:

To solve this technical problem, the invention teaches an isolated protein or peptide containing or consisting of one or more amino acid sequences SEQ ID 1 to 46. Protein or peptides according to the invention can be expressed in a cell, in particular a transformed cell, for biosynthesis, but it is also the import externally generated Protein or peptide in a biosynthetic exporting cell possible. In both cases, it is recommended that the expression of a corresponding natural gene of the cell is inhibited or reduced. The protein or peptide is preferably functional for a partial synthesis step of the biosynthesis of a lysolipin derivative, especially a lysolipin antibiotic.

The invention further relates to an isolated nucleic acid, in particular DNA, for example genomic DNA, cDNA, or RNA, in particular mRNA, coding for a protein or peptide according to the invention.

With the invention, the targeted molecular genetic manipulation of secondary metabolite biosynthetic gene clusters is made possible, whereby new, modified substance derivatives can be biologically produced. The invention in this context is based on the initial identification and characterization of a biosynthetic gene cluster for bacterial, N-containing

Xanthones. This provides the basis for any targeted genetic manipulation to produce lysolipin derivatives that are totally synthetic, if at all, very difficult to produce. These can in turn be semisynthetically derivatized as precursor molecules. In addition, the knowledge gained from the gene cluster sequence allows a rapid molecular genetic access to the biosynthesis of all other bacterial N-containing polycyclic xanthones with pharmaceutical potential.

The invention also encompasses homologs of the disclosed sequences wherein the homology is at least 60% identity, preferably greater than 80% or 90% identity, most preferably greater than 95% identity (calculated using the program MEGALIGN, DNASTAR LASERGENE, in U.S. Pat Registration date valid version). In the case of the nucleic acid sequences, complementary or allelic variants are also included. Furthermore, sequences are included which only partial sequences or gene fragments of explicitly disclosed sequences or complementary sequences thereof, with the proviso that in the case of the nucleic acids these partial sequences have a sufficient length for a hybridization with a nucleic acid according to the invention, at least 50 or 150 bases, and in the case of the proteins or peptides, optionally encoded by a nucleic acid binding with at least the same affinity to a protein- or peptide-specific target molecule. Furthermore, all nucleic acids which hybridize with nucleic acids used according to the invention are those which are stable under stringent conditions (5 ° C. to 25 ° C. below the melting temperature; see, in addition, JM Sambrook et al., A laboratory manual, CoId Spring Harbor Laboratory Press (1989). and EM Southern, J Mol Biol, 98: 503ff (1975)). It is understood that the invention also comprises expression cassettes, ie one or more of the nucleic acid sequences according to the invention having at least one control or regulatory sequence. Such an expression cassette may also comprise a sequence for a known protein, wherein in the course of translation, a fusion protein of a known protein and a protein or peptide according to the invention is formed. Likewise, antisense sequences to the above nucleic acid sequences are also included. In connection with uses according to the invention, the terms of the nucleic acids or proteins or peptides in addition to the full lengths of the disclosed sequences (see also the preceding paragraph) also include partial sequences or gene fragments thereof, with a minimum length of 21 nucleotides, preferably a minimum length of 30 to 90 Nucleotides, in the case of the nucleic acids and a minimum length of 7 amino acids, preferably a minimum length of 10 to 30 amino acids, in the case of proteins or peptides. In particular, between 21 and 90 as well as 7 and 30 every single numerical value is possible as a minimum length. Previously known sequences and / or their use, which fall under the definitions used in the context of this description, can be excluded by a disclaimer in claims.

The invention further comprises an isolated transformation vehicle, in particular plasmid or cosmid containing an inventive

Nucleic acid. It can contain a plurality, in particular 1 to 50, identical and / or different nucleic acids according to the invention. In this case, it is particularly recommended if the majority of the nucleic acids form a biosynthetic gene cluster or a part of a biosynthetic gene cluster for a lysolipin derivative. The transformation vehicle may additionally contain at least one regulatory nucleic acid sequence, wherein the nucleic acid according to the invention is under the control of the regulatory nucleic acid sequence, in particular of a promoter. Of course, one or more regulatory sequences can be set up with the proviso that a plurality of nucleic acids, in particular a gene cluster or a part of a gene cluster are controlled.

The invention further relates to a cell comprising a foreign nucleic acid according to the invention or several different such foreign nucleic acids and / or an inventive foreign protein or peptide or several different such foreign proteins or peptides, wherein the cell optionally with a foreign nucleic acid according to the invention or several different such foreign nucleic acids can be transformed. In this case, a desired derivatization of a lysolipin derivative naturally synthesized by the cell can be achieved by a foreign gene different from the defined partial synthesis step in a lysolipin biosynthetic gene cluster of the cell instead of or in addition to a natural corresponding gene for a defined partial synthesis step Defined Teilyntheseschhtt is introduced. This can be done by constructing and introducing the entire foreign or mutant lysolipin biosynthetic gene cluster or parts thereof. It is as it were a mutated biosynthesis tailored according to the desired derivatization. It is also possible to inhibit or mutate a gene naturally contained in the cell coding for a protein or peptide which is functional for a partial synthesis of the biosynthesis of the lysolipin or a lysolipin derivative, especially in whole or in part to delete. This can also be done for several such genes naturally contained in the cell.

With the invention it is achieved that, besides targeted molecular genetic interventions, it is also possible to generate new lysolipin derivatives by introducing heterologous modification genes, that is to say by "combinatorial biosynthesis".

Suitable cells are preferably selected from the group of actinomycetes, e.g. the streptomycetes, or enterobacteria, e.g. E. coli. It is understood that a cell according to the invention is a mutant or recombinant of known strains produced with a transformation vehicle according to the invention, and that the antibiotic which can be produced therewith is derivatized with respect to known compounds.

The invention also encompasses lysolipin derivatives, in particular lysolipin derivative antibiotics, which are obtainable by cultivating a cell according to the invention and, after cultivation, isolating the lysolipin derivative from the cell and / or from the cultivation supernatant. A lysolipin derivative of the invention is for the preparation of a pharmaceutical composition for the treatment of bacterial or viral infections, mucus,

Tumors and / or inflammatory diseases suitable. In this case, the biosynthesis can be mutated to the effect that the new Lysolipinderivat has improved efficacy over known drugs. However, it is also possible for a lysolipin derivative according to the invention to be used "only" as a replacement therapy in the case of resistance to a known active substance. Then there is the advantage that by means of the active ingredient according to the invention in the first place a therapy is possible. Especially desireable, however, are new lysolipin derivatives with reduced side effects. Finally, particularly effective production methods are possible with the invention. The invention further relates to a pharmaceutical composition comprising a lysolipin derivative according to the invention or a plurality of such lysolipin derivatives in a physiologically effective dose. In addition, it may contain galenic auxiliaries and / or carriers.

The galenic preparation of a pharmaceutical composition according to the invention can be carried out in the usual way. Suitable counterions for ionic compounds are, for example, Na ⁺ , K ⁺ , Li ⁺ or cyclohexylammonium. Suitable solid or liquid pharmaceutical preparation forms are, for example, granules, powders, dragees, tablets, (micro) capsules, suppositories, syrups, juices, suspensions, emulsions, ointments, drops or solutions for injection (iV, ip, im, sc) or nebulization (Aerosols), transdermal systems or other topical application, as well as preparations with protracted release of active ingredient, in the preparation of which conventional auxiliaries such as excipients, disintegrants, binders, coating, swelling or lubricants, flavorings, sweeteners and solubilizers, Find use. As excipients may be magnesium carbonate, titanium dioxide, lactose, mannitol and other sugars, talc, milk protein, gelatin, starch, cellulose and its derivatives, animal and vegetable oils such as cod liver oil, sunflower, peanut or sesame oil, polyethylene glycols and solvents, such as sterile water and monohydric or polyhydric alcohols, for example glycerol. A pharmaceutical composition according to the invention can be prepared by mixing at least one active ingredient used according to the invention in a defined dose with a pharmaceutically suitable and physiologically acceptable carrier and optionally further suitable active ingredients, additives or excipients with a defined inhibitor dose and preparing it to the desired dosage form. Preferably, a pharmaceutical composition according to the invention is galenically prepared for oral or parenteral administration.

Dosages are 0.01 to 5.0 mg / kg / day for a 75 kg adult, in particular 0.01 to 1, 0 mg / kg / day, (orally), 0.001 to 2.5 mg / kg / day, in particular 0.005 to 1, 0 mg / kg / day, (ip) in question. In the case of topical application, the active ingredient concentration may be 0.001 to 1.0% by weight, in particular 0.01 to 0.1% by weight, of the ointment.

In the following, the invention is explained in more detail by description of the identification, sequencing, annotation of the lysolipin biosynthetic gene cluster. In these embodiments, individual features of the invention may be implemented alone or in combination with other features. The particular embodiments described are merely illustrative and for a better understanding of the invention and are in no way limiting.

Example 1: Identification of the Lysolipin Biosynthetic Gene Cluster by Genetic Screening and Functional Detection by Heterologous

Expression of the entire gene cluster

The identification and analysis of all genes necessary for the biosynthesis of a bacterial natural product is made possible in the order of the Actinomycetes, to which the well-known genus of Streptomycetes belongs, by the fact that in general all the genes required for synthesis are physically adjacent to one another on the chromosome Gene clusters are organized. In addition to the genes that code for biosynthesis, these gene clusters also contain the genes necessary for regulation, export, and resistance mediation. This means that after the

Identification of a substance-specific biosynthetic gene; all remaining genes of the cluster can be identified by analysis of adjacent regions.

In recent years, the use of the strategy of "reverse genetics" has exploited the conservation of genes involved in biosynthesis certain conserved structural elements are found to be very efficient. This strategy first uses the chemical structure and results of in-situ studies to postulate a potential biosynthetic scheme. In the second step, characteristic genes or enzymes are determined which are conserved for the structural class to be identified. After sequence comparison, these highly conserved motifs are determined, which can be used to generate primer probes for PCR or hybridization. The targeted combination of different probes then leads to the discovery of genes or gene clusters that could be involved in the biosynthesis of the substance to be investigated.

The chemical structure of lysolipin and feeding experiments suggest that lysolipin is synthesized from 12 malonyl CoA units following a typical type II polyketide synthesis scheme. In contrast to complex polyketides (such as macrolides), which are synthesized by the modularly organized multifunctional type I polyketide synthases, the biosynthesis of the backbone of aromatic polyketides is iterative by the minimal PKSII, which consists of 3 separate enzymes: 2 ß-ketoacyl synthase subunits KSa and KSB (previously known as "Chain Length Factor", CLF) and the "Acyl Carrier Protein" (ACP) In the course of biosynthesis, the KSa and KSβ subunits are believed to control the selection and eventual processing of the starter ketide the number of condensations and the actual iterative condensation between the acyl thioesters and the growing C chain, which acts as a kind of anchor for the growing polyketide chain.

Comparing the three minimal PKS-encoding genes of different PKSII biosyntheses among themselves, it is striking that these are highly conserved and usually have the characteristic organization KSα-KSß-ACP. However, there are exceptions, such as in the daunorubicin biosynthetic gene cluster of S. peucetius, in whose gene cluster the ACP is separated from KSa and KSß. KSa or KSß are thus suitable marker genes for aromatic polyketides and the use of derived gene probes should therefore also in the case of lysolipin producers, for the identification of PKSII clusters ("reverse genetics"), leading to the biosynthesis of aromatic polyketides, such as lysolipin This strategy has already been successfully applied to the identification of different PKSII clusters (Metsä-Ketelä et al., 2002, Appl. Environ.Micorbiol., 68, 4472-4479), since many PKSII gene clusters are found in streptomycetes , which may be involved in the biosynthesis of spore pigments but also in the biosynthesis of several aromatic polyketides in a strain on the one hand, the use of a further gene probe for the specific identification of the lysolipin biosynthetic gene cluster is advantageous.

In the case of lysolipin, there is a strategy here in which the introduction of the Cl substituent at C1 in the center. The specific halogenation of natural products of bacterial origin, e.g. Chloro-tetracycline, pyrrolnitrin or vancomycin is catalyzed by NADH / FAD-dependent halogenases (van Pee, 2001, Arch. Microbiol. 175, 250-258). All halogenases that are involved in the halogenation of phenyl or pyrrole radicals, have high similarities at the protein level and allow the selection of highly conserved regions, which in turn are suitable for the derivation of primers. Such primers can be used for the identification of halogenase genes and associated biosynthetic gene clusters (Piraee and Vining, 2002, J. Ind., Microbiol., Biotechnol., 29, 1-5).

1.1.

Identification of the lysolipin biosynthetic gene cluster was done using PKSII and halogenase-specific gene probes against spotted clones of a cosmid library of strain CB28.

The method of gene bank production is based essentially on protocols described under Beye et al., 1998, GENOMICS 49, 317-320 and Burgtorf et al., 1998, GENOMICS 52 (2): 230-2. homogenized

Bacterial cultures were embedded in 0.5% low melting point agarose (SeaPlaque GTG, Biozyme) followed by 2 mg / ml HEW lysozyme (Roth) for 14 h at room temperature and 1 mg / ml proteinase K (Merck) for 24 h incubated at 50 ⁰ C. The embedded DNA was partially cleaved with Sau3A \, extracted with gelase (Epicentre) and dephosphorylated according to methods described. The genomic DNA was ligated to 750 ng of βamHI digested cosmid vector pOJ436 (Kieser et al., 2000, Practical Streptomyces Genetics, The John Innes Foundation, Norwich, England.), Desalted, packaged (Gigapack III Gold Packaging Extract, Stratagene) and into DH5α (Invitrogen) transfected. Cosmid clones were picked in 384 MTP followed by nylon filters

(Amersham) spotted. After the colonies had grown on the membranes, they were processed according to Nizetic et al., 1991, PNAS 88: 3233-7 and hybridized non-radioactively using the standard methods (Roche). To identify PKSII-encoding cosmids, a homologous probe was first prepared using the genomic DNA from CB28 (PCR primers, see Metsä-Ketelä et al., Appl. Environ.Micorbiol., 68, 4472-4479). The PCR mixture had the following composition: 1.0 μl of CB28 genomic DNA (about 0.2 μg), 2.5 μl of 10 × buffer, 2.5 μl of PCR-DIG Probe Synthesis Mix (Roche), 5.0 μl Q-Solution, 0.5 μl PKSII-FOR primer (50 pmol), 0.5 μl PKSII-REV primer (50 pmol), 0.5 μl Qiagen Taq polymerase (2.5 u), 14 , 5 μl H ₂ O. The PCR was carried out in a PCR instrument from MJ Research (PTC-225) under the following conditions: 1 × (2 min, 95 ° C.), 30 × (95 ° C., 1 min, 72 ° C, 2 min, 72 ° C, 1.30 min), 1 x (72 ° C, 5 min). PKSII amplicons show a characteristic size of about 600 bp. A use of the thus amplified homologous PKSII probe in a hybridization against the CB28

Cosmid library revealed 13 hybridizing cosmid clones that could be confirmed by a control PCR. In order to select from these cosmids those most likely to encode portions of or the entire lysolipin biosynthetic gene cluster, another probe directed against halogenase genes was used. For this purpose, the following conserved primers were also used with the help of the genomic DNA of CB28: Halo-For: GCG GCT GCA G (GC) T GG (AGT) (AT) (GC) AT (CT) C CG (CT) T Halo Rev: CC (GC) (GC) TG GAT CC (GC) CGGGTC (GC) A (GCT) GAA GC (see also: van Pee, Zehner, S., 2003. Enzymology and molecular genetics of biological halogenation In: Gribble.G. (Ed.), The Handbook of Environmental Chemistry, Part P Natural Production of Organohalogen Compounds, Springer Verlag, Heidelberg, Vol. The PCR was performed using the PCR DIG Probe

Synthesis Kit (Roche) and had the following composition: 1.0 μl CB28 genomic DNA (approximately 0.2 μg), 5 μl 10 × buffer, 5 μl PCR DIG labeling mix, 1 μl Halo-For- and Halo Rev primer (50 pmol), 0.75 μl enzyme mix, 36.25 μl H ₂ O. The PCR was performed in a MJ Research PCR apparatus (PTC-100) under the following conditions: 1 × (2 min, 94 ⁰ C), 25 x (94 ° C, 1 min; 61 ⁰ C, 1 min; 72 ° C, 1 min), 1 x (72 ° C, 10 min). Characteristic halogenase amplicons showed a size of about 260 bp. Use of the thus amplified homologous halogenase probe in a hybridization against the CB28 cosmid library yielded 11 hybridizing cosmid clones that could be confirmed by control hybridization. A total of 3 cosmid clones (28-4H04, 28-4022, and 28-3J01) cohybridized against PKSII and halogenase probes, so that they were likely to encode parts or the complete lysolipin biosynthetic gene cluster.

1.2.

Evidence that the identified co-hybridizing cosmid 28-4H04 encodes all genes necessary for the biosynthesis of lysolipin was made by heterologous expression in S. albus.

Aromatic polyketide biosynthetic gene clusters consist of about 30 to 40 ORFs having a coding capacity of between 30 to 35 kb. Since average insert sizes of about 40 kb are achieved in the generated cosmid library, there is the theoretical possibility that the complete lysolipin biosynthesis gene cluster is located on one of the 3 co-hybridizing cosmids. Functional detection may be by heterologous expression of the entire cluster and production of lysolipin in a foreign host respectively. Apart from S. coelicolor, S.albus J1074, which has been used successfully as a heterologous host for the production of rebeccamycin, among others, is a suitable host (Sanchez et al., 2002, Chem Biol. 9 (4): 519-31). To demonstrate the possible presence of the complete lysolipin biosynthetic gene cluster, the co-hybridizing cosmid 28-4H04 was transferred to S. albus. The transfer of genetic information to actinomycetes is possible using the intergeneric conjugation of E. coli (Kieser et al., 2000, Streptomyces Genetics, The John Innes Foundation, Norwich, England). A prerequisite here is an E. coli donor strain containing IncP plasmid, such as ET12567 (pUB307) [McNeil et al., 1992, Gene 111, 61-68], which simultaneously contains an oriT-carrying and therefore mobilizable plasmid, which can be mobilized to actinomycetes during conjugation by the IncP plasmid. The basic cosmid vector pOJ436 has a corresponding oriT and in addition an integrating function of the Actinomycetenphagen PhiC31, which allows the integration of the cosmid into the chromosome of a variety of Streptomyceten, including in S. albus (Kieser et al., 2000). The cosmid 28-4H04 was intergenerically conjugated to S. albus by E. coli (standard method see: Kieser et al., 2000). Successful transfer and integration of the cosmids can be detected by selection on the apramycin resistance gene marker aacC4. Therefore, the transconjugation approach was overlaid with 1 mg apramycin (selected for transfer and integration of the cosmid) and 1 mg phosphomycin (kills the unwanted E. coli donors present in the transconjugation mixture). After 4 - 6 days of incubation at 28 ⁰ C more transconjugants were visible, their apramycin resistance could be successfully verified.

In order to detect a potential heterologous lysolipin production in the transconjugant S. a / ιfc> ivs / 28-4H04, this was fermented under production conditions and analyzed after extraction with the aid of LC-DAD-MS. The negative control used was a S. albus culture without cosmid, which was cultivated and extracted under identical conditions. The cultivation took place with the following cultures. Preculture: 50 ml of R5 medium containing 25 μg / ml apramycin [composition R5 s. Kieser et al., 2000, pH 7.2] were inoculated with the transconjugant S. a / ö.vs / 28-4H04 and incubated for 48 h at 28 ° C and 160 rpm; Main culture: 100 ml of R5 medium containing 25 ug / ml apramycin [Composition R5 above] were treated with 1 ml of the preculture and h for 120 at 28 ⁰ C and incubated rpm 160th The control culture (S. albus) was cultured without the addition of apramycin.

The extraction was carried out as follows: i) each 1 ml of the main culture was extracted after addition of 50 .mu.l of 1 N HCl with 1 ml of ethyl acetate and the organic phase was concentrated to dryness, ii) the residue was taken up in 100 .mu.l of MeOH and the production of Lysolipin examined by LC-DAD-MS. The injection volume was 20 μl.

LC-DAD-MS analysis (instrument specifications: HPLC: Waters Alliance 2790; column: Grom Sil 120 ODS-4 HE, 3 μm, dim. 40 x 4 mm; precolumn: Phenomenex C18 ODS, 4 mm L x 3.0 mm ID; MS: Micromass Q-TOF 2) was carried out with the following gradient system (solvent: A - water with 0.1% formic acid, B - acetonitrile):

Time A% B% Flow Curve 0.00 98.0 2.0 1.000 1

5.00 16.7 83.3 1.000 6

6.00 4.0 96.0 1.700 6

8.00 4.0 96.0 1.700 6

8.50 98.0 2.0 1.000 6 10.00 98.0 2.0 1.000 6

The LC-DAD-MS analysis of the transconjugant S. aΛbt; s / 28-4H04 clearly shows that, in contrast to the negative control (S. albus), it synthesizes a substance whose retention time, UV spectrum and mass correspond to those of lysolipin I. , 2 to 5, the total ion current (TIC) chromatograms (positive mode, FIG Ion chromatograms (positive mode and between m / z 597 to 599

(Lysolipin I [M + H ⁺ ] ⁺ : 598); Fig. 3), DAD UV (Base Peak Index (BPI)) chromatograms, and the focused DAD UV chromatograms (BPI; 260-280 nm, Fig. 5) of the transconjugant S. a / jbαs / 28-4H04 (top) compared to the negative control S. albus (middle) and lysolipin I (bottom, pure lysolipin I in a biocatalysis approach). In the transconjugant chromatogram, an additional peak appears, which has a retention time of about 4.6 min. The substance corresponding to this peak shows a UV spectrum (Figure 6, top) which corresponds to the UV spectrum of lysolipin I (pure lysolipin I in a biocatalysis approach, Figure 6, bottom). A comparison of the heterologously synthesized substance at 4.6 min (Figure 7, top) with lysolipin (pure lysolipin I in a biocatalysis approach, Figure 7, bottom) shows that a substance was formed in the transconjugant, its mass and isotope distribution pattern which corresponds to the single-chlorinated lysolipin I.

Example 2. Sequencing of the complete biosynthetic gene cluster,

Sequence annotation and development of a biosynthetic scheme

The above successful heterologous expression showed that cosmid 28-4H04 carries all the genes necessary for the synthesis of lysolipin. to

Determination of the sequence of the putative lysolipin biosynthetic gene cluster, the approximately 43 kb insert of the cosmid was completely shotgun sequenced. Overall, a sequence of 43,202 bp double-stranded was determined, to which reference is made to the sequence listing (SEQ. ID 93). The GC content of the entire sequenced region was determined to be 72.2% for actinomycetes. To identify potential lysolipin biosynthesis genes, extensive ORF (Open Reading Frame) analyzes were performed using the ORF finder "zCurve" (Guo et al., 2003, Nucleic Acids Res. 31 (6): 1780-9) and BlastP analyzes carried out. A total of 46 putative ORFs could be identified, with 'ORF46 being incomplete (the characteristics and designations of ORFs are summarized in Figure 8). These ORFs are in 9 groups different transcription direction organized (Fig. 9). Due to the annotation, the lysolipin biosynthetic gene cluster should comprise 44 ORFs (L / p genes) with a coding range of about 38.6 kb. The gene product of the wm 5 'region, presumably outside of the cluster of localized ORFs 1, shows highest homology to a conserved hypothetical protein from S. coelicolor (75% identity), while in the 3' region of the cluster the incomplete 'ORF46 show highest similarity (86 % Identity) to the terminal protein TpgR2 from Streptomyces rochei. It is generally noteworthy that the products of a total of 16 lysolipin biosynthesis genes (LIpOII, LIpOIII, LIpZI, LIpB, LIpCI, LIpD, LIPE, LIPF, LIPCII, LIPCIII, LIpOVI, LIPMI, LIPMII, LIPQ, LIPZIII, LIPRIV, see FIG. 8) highest homologies to gene products of the biosyntheses of pradimicin, rubromycin or griseorhodin have (pradimicin-type antibiotics). These three substances are also polycyclic polyketides synthesized by Typll PKS. Although the spiroketal antibiotics rubromycin and griseorhodin are structurally different from lysolipin, the postulated hexacyclic biosynthesis intermediate pradimicin shows striking structural similarities to lyolipin (Li and Piel , 2002, Chem. & Biol. 9, 1017-1026). This could explain why especially the backbone synthesizing enzymes (eg the lysolipin minimal PKS LIpD-E, see below) have high homologies to each other. Another conspicuous feature is that, due to the annotation, 16 Llp gene products are most similar to enzymes involved in redox processes. This fact explains the highly oxidized nature of lysolipin. At present no other cluster is known which has more enzymes of this class.

In the following, the function of the individual lysolipin biosynthesis genes or gene products found on the basis of the protein sequence comparison is described in accordance with the postulated biosynthesis scheme. The biosynthesis of the lysolipin backbone is shown in FIG. A peculiarity of lysolipin is the use of a malonate unit as a starter unit which is incorporated in the reverse orientation (Bockholt et al., 1994, J. Org. Chem. 59, 2064-2069). The interpretation of the installation studies further suggest that the malonyl-CoA starter must be converted into malonamide-CoA, which results in the necessary introduction of the N-group into the heterocycle A of lysohpin. Within the cluster HpA is a gene whose gene product is highly similar to asparagine synthases / glutaminamido. These enzymes transfer amino groups from glutamine to aspartate, resulting in the synthesis of asparagine and glutamate. Similarly, LIpA was able to transfer the ammo group of glutamine to malonyl-CoA, resulting in the formation of malonamide-CoA substantiates the fact that LIPA has a 51% identity to the asparagine synthase TcsG of the oxytetracycline cluster. Also in tetracycline biosynthesis, malonamoyl CoA postulates a nitrogen-modified starter (Hunter and Hill, 1997, in Biotechnology of Antibiotics (Strohl, WR , ed., 2nd Ed, pp 659-682, Marcel Decker, Ine, New York) The Condensation of the Putative Malonamide-A This is coded by the three genes HpD, HpE and HpF. The derived gene products show the highest similarities (52, 69 and 78%) to the ACP and to the β-ketoacyl synthase subunits KSβ and KSa of the rubromycin biosynthetic gene cluster Since the KSβ subunit should have an influence on the chain length of the polyketide, it is not surprising that the highest homology to the KSß subunit of rubromycin biosynthesis is found at 78%, since rubromycin with 12 required condensations belongs to the largest polyketides (the biosynthesis of lysohpin requires 11 condensations) The biosynthesis of lysohpin requires several praaromatic deoxygenations, which may be initiated during the formation of the polyketide (Bockholt et al 1994, J Org Chem 59, 2064-2069) In lysolipin Biosynthesegencluster are present with HpZI, HpZIII and HpZIV three genes whose gene products supreme Ide Similarity to 3-oxoacyl-ACP reductases, which could be responsible for the reductions shown in Figure 10 These reductions are the prerequisite for the deoxygenation of certain keto groups The biosynthesis of an aromatic backbone is usually completed by means of cyclases or aromatases, wherein at least 2 cyclases are needed. In the lysolipin biosynthetic gene cluster, the genes // pC / - /// contain three genes whose gene products show the highest identity to cyclases of griseorhodin / rubromycin biosynthesis. The highest identity of LIpCl-III especially to cyclases involved in the synthesis of pradimicin-like antibiotics underscores the fact that these antibiotics form a lysolipin-like intermediate.

Tailoring reactions are described below.

Oxidoreductions are shown in FIG. Since 9 of the 12 oxygen atoms present in lysolipin X (Figure 1) are from molecular oxygen, the presence of a large number of oxygen-introducing oxidoreductases is not surprising. The oxygenoredases belonging to the oxidoreductases catalyze the introduction of one (monooxygenase) or two oxygen atoms (dioxygenases) into the corresponding substrate. These can catalyze various chemical reactions, such as hydroxylation, epoxidation, and oxidative rearrangements, such as the Baeyer-Villiger reaction. They differ in their cofactors. Cytochrome P-450 monooxygenases (CYP450) and FAD-dependent mono- and dioxygenases are common in PKS biosynthesis (Rix et al., 2002, Nat. Prod. Rep. 19, 542-580).

Due to the sequence annotation with LIpOI, LIpOV and LIpOVIII, three FAD-dependent mono- or dioxygenases are present in the lysolipin biosynthetic gene cluster. The deduced gene products have a size of 461, 397 and 541 amino acids (AA). Due to the protein sequence and homology, it is currently not possible to predict at which specific oxygenation the respective FAD-dependent oxygenase is involved. Nevertheless, one of these enzymes is likely to be involved in the formation of xanthone or in the oxidative ring cleavage, since such Baeyer-Villiger oxidation in mithramycin biosynthesis is catalyzed by the FAD-dependent monooxygenase MtmOIV (Rodriguez et al. J Bacteriol 185 (13): 3962-5). Interestingly, however, is the fact that LIpOVIII highest similarity (47% identity) to Tetracenomycin hydroxylase TcmG, which is responsible for a 3-fold hydroxylation (Rafanan et al., 2000, Org Lett. 5, 2 (20): 3225-7). In addition, the cluster contains 3 genes HpOIV, HpOVI and HpOVII, whose derived gene products have the highest identity to cytochrome P450-dependent monooxygenases (CYP450). LIpOIV shows the highest similarity to MycG (48% identity), a CYP450, which in the course of the

Mycinamycin biosynthesis is responsible for both hydroxylation and epoxidation (Inouye et al., 1994 Mol Gen Gen. 245 (4): 456-64). As with some other CYP450 involved in the biosynthesis of secondary metabolites, downstream of HpOIV with HpK is a gene coding for a ferrodoxin. LIpOVI is most similar to a CYP450 from the pradimicin biosynthetic gene cluster. The derived gene products LIpOII and LIpOIII also show the highest similarity to genes of the pradimicin group (RubT, GrhV, GrhU), for which no function is described. A motif search (Pfam analysis), however, gives a clear "antibiotic biosynthesis monooxygenase" motif for both enzymes, so that both of them

Enzymes should be involved in oxygenation reactions. Furthermore, HpL encodes a gene product that has the highest homology to the hydroxylase annotated enzyme SnoaW. The exact function of this enzyme within the lysolipin biosynthesis remains to be clarified as well as the function of all other lysolipin oxygenases.

In addition, the following additional genes are localized in the cluster whose gene products show the highest similarity to oxidoreductases: LIPU: gene product has "alcohol dehydrogenase" motif and shows highest similarity to dehydrogenases.

LIpZII: Gene product shows highest identity (42%) to MmcJ, a F420-dependent tetrahydromethanopterin (H4MPT) reductase of mitomycin biosynthesis. This enzyme catalyzes carbonyl reduction and may be involved in the biosynthesis of the lysolipin starter. LIPS: Gene product shows highest identity (37%) to 3-hydroxybutyrate

Dehydrogenases involved in lipid metabolism.

A possible function of the two dehydrogenases, LIPU or LIPS, could be the oxidation of a lysolipin precursor necessary to introduce the methylene group (Figure 12).

The gene HpH encodes a gene product that shows the highest similarity to NADH / FAD-dependent halogenases (37-39%). Thus, LIpH will be responsible for the halogenation at C1.

The biosynthesis of lysolipin requires 4 O-methylations and N-methylation. In the cluster, 6 genes can be identified (HpMI to HpMVI) whose gene products have the highest similarity to O-methyltransferases of different antibiotic biosyntheses, such as tetracenomycin, griseorhodin and enterocin. Remarkably, the cluster codes for 6 O-methyltransferases, although only 5 are needed.

The cluster contains 4 regulatory genes (llpRI-llpRIV). LIPRIV has high homology (39%) to the RubS regulator of rubromycin biosynthesis, known as SARPs ("Streptomyces antibiotic regulatory protein"), a biosynthesis-specific transcriptional activator of some of the lysolipin genes LIpRI shows homology to a transcriptional regulator (presumably a repressor) and could mark the beginning of the cluster LIpRII and LIpRIII show the highest similarity to transcriptional activators (see Table 1).

The cluster hosts // pΛ / a gene whose deduced gene product shows the highest identity (30%) to a "multidrug transporter" from Lactococcus lactis, a protein likely involved in resistance mediation LIPQ, 2 gene products showing the highest identity (40-50%) to the RubQ and GrhM membrane proteins of rubromycin and griseorhodin synthesis, respectively. A function of these proteins has not previously been described. Nevertheless, both membrane proteins could also contribute to the mediation of resistance. Surprisingly, in the lysolipin gene cluster with HpG, one gene is located whose derived gene product has significant identity (42%)

Glycosyltransferases shows. There are kigamycin very well glycosylated lysolipin-like xanthones (Kunimoto et al., 2003, 56, 1012-1017), but glycosylated lysolipin derivatives are not yet known. Whether, as in the case of macrolides, a possible intermediate glycosylation is a resistance mechanism remains to be tested.

In the cluster with HpT and HpV are still 2 genes whose gene products have the highest homology to a polyketide synthase CurD annotated sequence or to WhiE. However, the function within the synthesis is unpredictable due to these homologies. Other genes / ORFs of unknown function are HpX, HpY, HpP and // pJ

For the sequence listing, the following gene list results (ID nucleic acid / ID

Protein): ORF1 47/1; HpX 48/2; HpY 49/3; HpP 50/4; MpRI 51/5; HpOI 52/6; HJ 53/7; HpOII

54/8; HpOIII 55/9; HpZI 56/10; HpB 57/11; HpCI 58/12; NpD 59/13; HpE 60/14; HPF

61/15; HpCII 62/16; HpCIII 63/17; HpU 64/18; HpG 65/19; MpA 66/20; HpRII 67/21;

HpOIV 68/22; HpK 69/23; HpT 70/24; HpL 71/25; HpOV 72/26; HpN 73/27; llpRIII

74/28; MpOVI 75/29; MpMI 76/30; HpOVII 77/31; HpMIl 78/32; llpOVIII 79/33; HpMIII 80/34; HpQ 81/35; HpZII 82/36; MpMIV 83/37; MpS 84/38; MpV 85/39; HpZIII

86/40; HpH 87/41; HpRIV 88/42; HpZIV 89/43; HPMV 90/44; MpMVI 91/45; and

ORF46 92/46.

SEQ. ID 93 is the total nucleic acid sequence.

Claims

claims:

1. Isolated protein or peptide comprising, in particular consisting of, one or more of the amino acid sequences SEQ ID 1 to 46.

2. Protein or peptide according to claim 1, wherein the protein or peptide for a partial synthesis step of the biosynthesis of a lysolipin derivative, in particular a lysolipine derivative antibiotic, is functional.

3. Isolated nucleic acid, in particular DNA, for example genomic DNA, cDNA, or RNA, in particular mRNA, coding for a protein or peptide according to one of claims 1 or 2, preferably containing or consisting of one or more of the

Nucleic acid sequences SEQ ID Nos. 47 to 92, or SEQ ID NOS 93.

4. Isolated transformation vehicle, in particular plasmid or cosmid, containing a nucleic acid according to claim 3.

5. An isolated transformation vehicle according to claim 4, containing a

A plurality, in particular 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, up to 46, of different nucleic acids according to claim third

6. Isolated transformation vehicle according to claim 4 or 5, additionally containing at least one regulatory nucleic acid sequence, wherein the nucleic acid or the nucleic acids under the control of the regulatory nucleic acid sequence, in particular a promoter, stands or stands.

7. Cell comprising a foreign nucleic acid or several different foreign nucleic acids according to claim 3 and / or a foreign protein or peptide or several different foreign proteins or peptides according to claim 1 or 2, which optionally with a foreign nucleic acid or several different foreign nucleic acids Claim 3 is transformed.

A cell according to claim 7 which is obtainable by transformation with a transformation vehicle according to any one of claims 4 to 6.

9. Cell according to claim 7 or 8, wherein a naturally contained gene coding for a protein or peptide, which is functional for a partial synthesis step of the biosynthesis of lysolipin or a lysolipin derivative, in particular a lysolipine derivative antibiotic, inhibited or mutated, in particular in whole or in part deleted, or being several naturally contained ones

Genes are inhibited or mutated, especially deleted.

10. A cell according to any one of claims 7 to 9, selected from the group of actinomycetes or enterobacteria.

1 1. A method for producing a cell according to any one of claims 7 to 10, wherein the cell is transformed with a transformation vehicle according to one of claims 4 to 6.

12. lysolipin derivative, in particular lysolipine derivative antibiotic, obtainable by culturing a cell according to any one of claims 7 to 10, and that after cultivation, the lysolipin derivative is isolated from the cell and / or from the cultivation supernatant.

13. A process for producing a Lysolipindehvates, in particular a Lysolipinderivat antibiotic, wherein a cell is cultured according to any one of claims 7 to 10, and wherein after cultivation, the Lysolipinderivat from the cell and / or from the culture supernatant is isolated.

14. Use of a Lysolipinderivates according to claim 12 for the preparation of a pharmaceutical composition for the treatment and / or

Prophylaxis of bacterial and / or viral infections, mucus, tumors and / or inflammatory diseases.

15. A pharmaceutical composition comprising a lysolipin derivative or several different lysolipin derivatives according to claim 12 in a physiologically effective dose.

16. Pharmaceutical composition according to claim 15, additionally containing galenic auxiliaries and / or carriers.

17. A pharmaceutical composition according to claim 15 or 16, galenically prepared for topical, oral or parenteral administration.

18. A process for the preparation of a pharmaceutical composition according to any one of claims 15 to 17, wherein a Lysolipinderivat or more lysolipin derivatives according to claim 12 mixed in physiologically effective dose with pharmaceutical excipients and / or carriers and galenically prepared, in particular for oral or parenteral administration ,